This invention pertains to the interaction between nucleic acids and 1,3,5-triazine derivatives, their use as inhibitors of the replication of the Hepatitis B virus (HBV), and their use in the treatment of viral hepatitis caused by HBV.
There is a great medical need for novel therapeutic drugs to treat viral infections. Approximately 300,000 Americans become infected annually with hepatitis B virus (HBV). Currently available drugs for the treatment of HBV have limited efficacy and have not exhibited lasting effects, such that virus titres rapidly increase following the termination of drug treatment. In addition, some classes of drugs, such as nucleoside analogs that inhibit the viral polymerase, often become ineffective due to the rapid appearance of resistant viral strains. For these reasons, it has become apparent that combination therapies utilizing multiple drugs that function by different mechanisms are likely to be more successful in the treatment of viral infections.
Small molecules can bind RNA with high affinity and specificity and can block essential functions of the bound RNA. Examples of such molecules are antibiotics such as erythromycin and aminoglycosides. The first suggestion that some antibiotic translation inhibitors interact specifically with RNA came from the genetic mapping of resistance to kanamycin and gentamicin to the methylation of 16S RNA (Thompson et al., Mol. Gen. Genet. 201:168, 1985). Erythromycin binds to bacterial RNA and releases peptidyl-tRNA and mRNA (Menninger et al., Mol. Gen. Genet. 243:225, 1994). 2-DOS-containing aminoglycosides bind specifically to the structures of HIV RNA known as the RRE, block binding of the HIV Rev protein to this RNA, and thereby inhibit HIV replication in tissue culture cells (Zapp et al., Cell 74:969, 1993). In addition, although aminoglycosides have long been developed as translation inhibitors, they were only recently shown to bind to rRNA in the absence of proteins (Purohit and Stem, Nature 370:659, 1994). Hygromycin B inhibits coronaviral RNA synthesis and is thought to do so by binding to the viral RNA and blocking specifically the translation of viral RNA (Macintyre et al., Antimicrob. Agents Chemother. 35:2630, 1991). Therefore, compounds that bind to functionally important regions of nucleic acids of viruses and microorganisms may be useful as inhibitors of replication or other functions, i.e., as antiviral agents and antibiotics.
The present invention pertains specifically to a novel class of drugs comprising RNA ligands that alter the function(s) of their target RNAs. This class of compounds comprises substituted 1,3,5-triazine derivatives that specifically recognize an essential and multifunctional RNA structure of the HBV pregenomic RNA known as the encapsidation signal (xcex5RNA). It has been unexpectedly found that this class of compounds can function as inhibitors of HBV replication. xcex5RNA, shown in FIG. 255A, consists of a short sequence that folds into a stem-loop structure interrupted by a 6 nucleotide bulge. xcex5RNA, which is contained within the open reading frame encoding the HBV precore protein, is also required for various steps of the HBV viral replication cycle, including encapsidation of the pregenome into viral particles and initiation of minus strand DNA synthesis. xcex5RNA may also play a role in folding and activation of the HBV-encoded polymerase.
Derivatives of melamine, 1,3,5-triazine-2,4,6-triamine, have been reported in the literature as suitable for various uses. For example, 2,4,6-tris(dimethylamino)-1,3,5-triazine is an antitumor agent known as Altretamine(copyright), used in the treatment of ovarian cancer (Cancer 71; 4 Suppl.: 1559, 1993). Similarly, Larvadex (N-cyclopropyl-1,3,5-triazine-2,4,6-triamine) has been used as an additive to animal feed stock to control house fly infestation in poultry houses (Poult. Sci. 62(12): 2371, 1983).
Further, Patel et al. (J. Inst. Chemists (India), 57, 1985) report a number of derivatives of 2-aryl amino-4-(4-methoxy anilino)-6-(4-chlorophenyl/phenyl hydrazido)-1,3,5-triazine having anti-bacterial activity, without data in support of this conclusion and absent any suggestion that the compounds could be used as antiviral agents.
U.S. Pat. No. 5,225,405 to Paramelle et al. refers to 4,6-bis-allylamino-1,3,5-triazin-2-yl derivatives which reverse acquired resistance to anti-cancer and anti-malarial agents. Paramelle et al. state that the disclosed triazine derivatives, when administered at the same time with a cytotoxic agent, reduce or completely suppress multidrug resistance. The triazine compounds presumably act by inhibiting the action of an inducible membrane protein that normally functions to increase the efflux of the cytotoxic agent, thereby reducing its intracellular concentration. Paramelle et al. are silent as to the use of the triazine derivatives as antiviral agents.
U.S. Pat. No. 4,508,898 to Ogilvie relates to nucleoside analogs that have a 1,3,5-triazine moiety, wherein the analog compounds exhibited antiviral activity. The compounds of Ogilvie, however, do not comprise 2,4,6-triamino-1,3,5-triazine derivatives, but rather, are N-substituted purine and pyrimidine compounds.
European Patent Application No. 172 608 to Kim et al. relates to 1,3,5-triazine derivatives that exhibit anti-ulcer, anti-inflammatory and anti-depressant activities. However, Kim et al. fail to suggest that the disclosed triazine derivatives can be used as antiviral agents.
Golankiewicz, et al. (J. Med. Chem. 38: 3558, 1995) report the isolation of several 1,3,5-triazine derivatives having antiviral activity. However, the derivatives were limited to imidazo-[1,5-xcex1]-1,3,5-triazine derivatives, with special emphasis on thio- and benzyl-substituted derivatives.
Kreutzberger et al. (Arzneim.-Forsch./Drug. Res. 36 (I)(4): 626, 1986) relates to aliphatically substituted chlorodihexylamino-1,3,5-triazines having antiviral activity. However, these compounds are structurally different from the compounds of the present invention.
WO 97/20825 discloses the isolation of various 1,3,5-triazine derivatives structurally distinct from those of the present invention. There is no suggestion that the triazine derivatives can be used as antiviral agents, but rather, that the compounds have utility as herbicides, insecticides, miticides, and bactericides.
U.S. Pat. No. 4,254,122 to Brown relates to 6-acylaminotetrahydro-1,3,5-triazine-2,4-dione derivatives that exhibit analgesic activities. The disclosed uses of the compound include their use as antiinflammatory agents and as inhibitors of prostaglandin synthetase.
Further, European Patent Application No. 795 549 to Gluzman et al. refers to bis-aryloxy(amino)-triazinyl-oxy(amino)aryl derivatives as antiviral agents. However, unlike the compounds of the present invention, the compounds of Gluzman et al. are dimers, linked by bicyclic or heterocyclic substituted moieties, and Gluzman et al. fails to suggest the use of the monomers as therapeutic compounds and/or compositions.
Thus, there is a need in the art for the identification of compounds that bind to functional viral nucleic acids, thereby inhibiting viral replication, and for specific antiviral agents that inhibit the replication of Hepatitis B virus. Such antiviral agents would be useful in the treatment of viral hepatitis caused by HBV.
The present invention provides methods for inhibiting viral and/or microbial replication, preventing or treating viral and/or microbial infection, and pharmaceutical formulations for use in such methods comprising a compound of the formulae IA 
or IB 
wherein R1, R2, R3, R4, R5, R6, R7 and R8 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, non-aromatic heterocyclic, fused or polycyclic ring, and aryloxy;
wherein said alkyl, alkenyl or alkynyl is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl or alkenyl; and
wherein said aryl, aryloxy, heteroaryl, non-aromatic heterocyclic, or fused or polycyclic ring is optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl or alkynyl;
or wherein R1 and R2 together, R3 and R4 together, or R5 and R6 together, optionally form a cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, heteroaryl, or fused or polycyclic ring, said cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, heteroaryl, or fused or polycyclic ring optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl and alkynyl;
or wherein R7 and R8 together optionally form a cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, or fused or polycyclic ring wherein said cycloalkyl, cycloalkenyl, non-aromatic heterocyclic and fused or polycyclic ring are optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl and alkynyl, with the proviso that when R7 and R8 together form a fused or polycyclic ring, the moiety of the fused or polycyclic ring that binds with N is non-aromatic;
and pharmaceutically acceptable salts thereof;
and a pharmaceutically acceptable carrier or diluent.
Further, it is an object of the present invention to provide antiviral and antibiotic formulations comprising one or more compounds represented by the formulae set forth above and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or diluent, and methods of administering such formulations to a patient in need of antiviral and/or antibacterial therapy. It is also an object of the present invention to provide a method of detecting a target nucleic acid by contacting the target nucleic acid with at least one compound of the formulae set forth above and pharmaceutically acceptable salts thereof, and monitoring an interaction between the target nucleic acid and the at least one compound of the formulae set forth above.